This grant will investigate the role of acid-base interactions in the regulated storage and release of granule contents in pancreatic acinar cells. We will test the hypothesis that formation of GP2 tetramers in the acidic milieu of the trans-Golgi network (TCN) organizes a GP2/proteoglycan (PG) matrix tightly associated with the lumenal surface of zymogen granule (ZG) membranes and that this matrix functions in (a) membrane sorting during granule assembly in the TCN, (b) inactivation of ZG membranes during the storage phase of secretion, and (c) regulated trafficking of ZG membranes from the apical plasma membrane (APM) after exocytosis. Since the acidic pH of the TGN plays a critical role in condensation of secretory proteins, alkalinization of the acinar lumen appears to be required for (i) neutralization of the acidic pH of exocytic contents and (ii) solubilization of aggregated (pro)enzymes. We will determine if alkalinization of the acinar lumen leads to the release of the GP2/PG matrix from the APM and if pH-dependent matrix removal regulates internalization of ZG membranes for reuse during the secretory process. Taken together, our preliminary findings suggest that lumenal factors including acid-base interactions and matrix assembly and disassembly processes perform critical functions during regulated storage and release of pancreatic enzymes. Definitive analysis of GP2 function in regulated pancreatic secretion will be made by constructing a GP2 null mutation in J1 embryonic stem cells and introducing this trait into the germ line of transgenic mice. Analysis of zymogen granule structure and function and regulated secretory performance in normal and mutant mice will allow us to confirm the cellular function(s) of GP2, the major glycoprotein in ZG membranes. These studies can be expected to provide valuable information concerning a recently described gene family (GP2/THP), which encodes a new class of GPI-anchored proteins targeted to intracellular secretory compartments in polarized epithelial cells. A detailed understanding of lumenal processes including acid-base interactions and matrix assembly and disassembly processes in trafficking of ZG membranes at the APM provides, for the first time, important insights into pancreatic dysfunctions observed in these processes (dramatic loss of zymogen granules and marked dilatation of the acinar lumen) in patients with cystic fibrosis.